Summary of work: The mitochondrial DNA (mtDNA) accumulates high levels of oxidative damage due to its proximity to the electron transport chain, where most reactive oxygen species are generated. Oxidative damage can cause mutations, deletions and lead to cell death. 8-hydroxyguanine (8-oxoG), a major oxidative DNA lesion, accumulates with age in the mtDNA. Repair of oxidative DNA damage is carried out by the base excision repair (BER) pathway. We investigated the repair mechanisms for the removal of DNA lesions, particularly oxidized bases, from the mitochondrial genome. We have explored the role of the oxoguanine DNA glycosylase (OGG1) in mtDNA repair and find that OGG1 plays a crucial role in the repair of oxidized lesions in mitochondria and is probably the only DNA glycosylase for 8-oxoG removal in these organelles. Liver mitochondrial DNA from OGG1-/- mice accumulates high levels of 8-oxoG lesions. We measured respiratory activities in liver mitochondria isolated from these animals to test the hypothesis that high levels of oxidative DNA damage cause mitochondrial dysfunction. We find that mitochondria respiration, as well as respiratory complexes activity is similar in mitochondria isolated from knockout mice to that of wild type mice. We concluded that the levels of 8-oxoG found in these animals are not enough to directly cause mitochondrial respiratory dysfunction. In human cells two distinct OGG1 isoforms are expressed, alpha and beta. Beta-OGG1 localizes exclusively to mitochondria and was believed to provide the 8-oxoG glcycosylase activity. We purified recombinant b-OGG1 and found that the protein lacks glycosylase activity. Site-directed mutagenesis studies identified two aminoacids that are found in the b-isoform that render the a-isoform inactive. We also found that approximately 10% of a-OGG1 localizes to mitochondria and may provide the 8-oxoG glycosylase activity. NTH is the other major glycosylase for repair of oxidative DNA damage. All BER enzymes are encoded in the nucleus and transported to mitochondria; however there is very limited information on the regulation of mitochondrial BER. We measured BER activities in mitochondria that lack mtDNA (rho-). Despite the absence of mtDNA a complete set of BER enzymes was present in mitochondria, and most activities were only slightly decreased compared to wt mitochondria. Interestingly, nuclear BER activities were also affected by the absence of mtDNA, suggesting an interesting cross-talk between BER in both compartments. Mitochondria are comprised of two membranes (outer and inner) enclosing an aqueous matrix compartment. We studied the spatial organization of BER in mitochondria and find that most BER activities are not freely soluble in the matrix, but rather associated with the membrane fraction. This association is likely electrostatic in nature, as it can be disrupted by high salt concentration. The existence of this ?higher order DNA repair complex? has profound implications for mtDNA repair, as it suggests a mechanism in which the DNA flows through this stationary complex. We are now investigating whether mammalian mitochondria have other repair pathways that operate in the nucleus, such as mismatch repair. Our results show that human mitochondria contain a mismatch binding activity, and we are now identifying this protein. These observations, along with results from others clearly establish that mammalian mitochondria have a functional mismatch repair pathway. One strong point of our studies is that we assay for DNA repair activity and measure the actual occurrence of the lesions in DNA using HPLC and other analytical techniques. We analyzed the levels of 8-oxoG and other oxidized bases in mouse liver DNA and found that the levels of the ring-opened oxidative lesion fapyguanine is significantly higher than that of 8-oxoG. We find that fapyguanine, and fapyadenine are repaired by the same set of DNA glycosylases that remove 8-oxoG and thymine glycols from DNA, at similar or higher efficiencies. These results indicate that the accumulation of these lesions may have important biological consequences, at least as relevant as those of 8-oxoG.